Systematic Functional Interrogation of Rare Cancer Variants Identifies Oncogenic Alleles

Eejung Kim, Nina Ilic, Yashaswi Shrestha, Lihua Zou, Atanas Kamburov, Cong Zhu, Xiaoping Yang, Rakela Lubonja, Nancy Tran, Cindy Nguyen, Michael S Lawrence, Federica Piccioni, Mukta Bagul, John G Doench, Candace R Chouinard, Xiaoyun Wu, Larson Hogstrom, Ted Natoli, Pablo Tamayo, Heiko Horn, Steven M Corsello, Kasper Lage, David E Root, Aravind Subramanian, Todd R Golub, Gad Getz, Jesse S Boehm, William C Hahn

Cancer genome characterization efforts now provide an initial view of the somatic alterations in primary tumors. However, most point mutations occur at low frequency and the functional consequences of these alleles remain undefined. Here we have developed a scalable systematic approach to interrogate the function of cancer-associated gene variants. Specifically, we subjected 474 mutant alleles curated from 5,338 tumors to pooled in vivo tumor formation assays and gene expression profiling. We identified 12 transforming alleles including two in genes (PIK3CB, POT1) that have not been previously shown to be tumorigenic. One rare KRAS allele, D33E, constitutively activates RAS effector pathways and is in spatial proximity to the positions of common oncogenic KRAS mutants. By comparing gene expression changes induced upon expression of wild type and mutant alleles, we inferred the activity of specific alleles. The observation that several alleles found to be mutated only once in 5,338 tumors rendered cells tumorigenic demonstrates the importance of integrating genomic information with functional studies to expedite the interpretation of cancer genomes.